Project Summary The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) harbors the epitopes of broadly neutralizing antibodies (bNAbs) and is the main target of HIV-1 vaccine development. High-resolution structures of BG505 SOSIP.664 trimers have provided a rational basis for vaccine design. Although current gp140 designs can produce native-like trimers for selected strains after optimization, the underlying causes of Env metastability are still unknown and a general design platform applicable to all HIV-1 strains and subtypes remains an unmet challenge. Furthermore, despite the increasing appreciation for the advantages of virus-like particles (VLPs) in bNAb elicitation, nanoparticulate display of native-like trimers has not been rigorously explored in HIV-1 vaccine development. In this R01, we will develop HIV-1 vaccine candidates by combining trimer design, crystallography, electron microscopy (EM), nanoparticle display, immunization, serological assays, antibody isolation, repertoire sequencing, and antibody lineage analysis. In Aim 1, we will investigate the fundamental causes of HIV-1 Env metastability and develop the uncleaved, prefusion-optimized (UFO) trimer platform. We hypothesize that gp41, which contains a structurally strained heptad repeat 1 (HR1) with a disordered N-terminal bend (N-HR1), is the source of metastability. We have designed 1st-generation UFO trimers with a modified N-HR1 and solved their structures. We will develop an optimized UFO platform applicable to diverse Envs with high purity, yield, and stability and select representative UFO trimers for vaccine development. We will assess trimer antigenicity using a panel of bNAbs and non-NAbs and determine their atomic structures. In Aim 2, we will engineer stable gp140 trimer-presenting nanoparticles as vaccine candidates. We hypothesize that self-assembling nanoparticles with underlying 3-fold axes can be used to display native-like gp140 trimers. We have reported gp140 nanoparticles based on the 24-mer ferritin (12 nm) and 60-mer E2p (23nm). We will optimize UFO gp140 nanoparticles based on ferritin, E2p, and a hyperstable 60-mer, I3-01 (25nm) for in vivo testing in Aim 3. We will validate nanoparticle assembly by EM and evaluate nanoparticle antigenicity using a panel of antibodies. In Aim 3, we will assess the immunogenicity and B-cell response for a subset of optimized UFO trimers and nanoparticles in WT mice, rabbits, and nonhuman primates (NHPs). We hypothesize that UFO trimers can induce tier-2 NAbs in all animal models, while gp140 nanoparticles displaying 8-20 UFO trimers can elicit tier-2 NAbs more effectively than trimers alone. We will first screen UFO trimers and nanoparticles in WT mice and assess serum neutralization using purified IgGs. We will select a small set of immunogens for rabbit immunization and advance the most promising ones to NHPs. In our preliminary study, gp140 nanoparticles induced autologous tier-2 NAbs to BG505.T332N in mice and rabbits within 6-8 weeks, supporting our approach. We will isolate vaccine-elicited tier-2 NAbs for functional and structural studies and perform next-generation sequencing (NGS) to study B-cell repertoires and trace NAb lineage development. Our goal is to identify the most effective vaccine candidates for future clinical studies.